WO2016172979A1 - 一种低致敏低苦味大豆低聚肽及其制备方法和应用 - Google Patents

一种低致敏低苦味大豆低聚肽及其制备方法和应用 Download PDF

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WO2016172979A1
WO2016172979A1 PCT/CN2015/078137 CN2015078137W WO2016172979A1 WO 2016172979 A1 WO2016172979 A1 WO 2016172979A1 CN 2015078137 W CN2015078137 W CN 2015078137W WO 2016172979 A1 WO2016172979 A1 WO 2016172979A1
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low
soybean
enzymatic
preparation
sensitization
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PCT/CN2015/078137
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English (en)
French (fr)
Inventor
蔡木易
谷瑞增
鲁军
马涛
潘兴昌
董哲
马勇
徐亚光
马永庆
金振涛
陈亮
陆路
刘文颖
魏颖
张海欣
刘艳
曹珂璐
王憬
李国明
周明
陈辉
李佳霁
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中国食品发酵工业研究院
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Application filed by 中国食品发酵工业研究院 filed Critical 中国食品发酵工业研究院
Priority to PCT/CN2015/078137 priority Critical patent/WO2016172979A1/zh
Priority to JP2017516291A priority patent/JP6505211B2/ja
Priority to CN201580015309.0A priority patent/CN106455625B/zh
Publication of WO2016172979A1 publication Critical patent/WO2016172979A1/zh
Priority to US15/424,715 priority patent/US20170143001A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24028—Bacillolysin (3.4.24.28)

Definitions

  • the invention relates to a soybean oligopeptide, in particular to a low sensitization and low bitterness soybean oligopeptide and a preparation method and application thereof.
  • Soy protein is a kind of vegetable protein. Its amino acid composition is similar to that of milk protein. The content of various essential amino acids is rich. It can be equal to animal protein in nutritional value. It is also the closest to human amino acid in gene structure. It is the most nutritious. Plant protein. However, there are various allergens in soy protein, such as glycinin, ⁇ -conglycinin, P34, GlymBd 28K, etc., wherein glycinin and ⁇ -conglycinin are the main constituents of the protein in soybean, About 70%; while some soy proteins such as soybean trypsin inhibitor (STI) remain stable in conventional production processes (such as high temperature conditions) and are often used as indicators to detect soy allergenic proteins. At present, about 1 to 6% of babies are affected by soy allergens, which cause respiratory allergic reactions such as respiratory, skin and gastrointestinal symptoms, and with the increasing number of soybean products, the incidence of adult allergies in adults It is also rising.
  • soy allergens such as glycinin,
  • Methods for desensitizing soy protein include heat treatment, chemical treatment, fermentation, enzymatic, and the like.
  • Heat treatment is the most commonly used method for desensitization of soy allergens, which can change the structure of soy protein and reduce the sensitizing activity of antigenic proteins.
  • chemical treatment mainly uses chemical reagents to reduce the activity of trypsin inhibitors, but it inevitably causes food safety problems such as chemical residues.
  • the fermentation method mainly uses microorganisms such as mold and Bacillus subtilis to degrade the antigenic protein in soybean products.
  • microorganisms such as mold and Bacillus subtilis to degrade the antigenic protein in soybean products.
  • fermentation can hydrolyze soy protein into small peptides with low sensitization, whether the hydrolyzed protein retains the necessary conformation recognized by the antibody Still a problem.
  • the publication No. CN101990984A discloses a method for preparing fermented soybean meal with high anti-oxidation and low sensitization for feeding.
  • the fermentation of the soybean meal fermentation base is carried out by using Aspergillus oryzae, although the macromolecular protein is obviously degraded after fermentation, but it is not
  • the sensitization of the fermented product was examined, so that it was not possible to determine whether the sensitized soybean fragment was still present in the fermented product; in addition, the method did not evaluate the taste of the fermented product.
  • Herian et al. used the allergen adsorption assay (RAST) to detect the sensitization of five traditional soybean fermented products, including bean sprouts, acid-hydrolyzed soy sauce, mold-hydrolyzed soy sauce, soybean meal and bean paste, and the results showed five soybeans.
  • the ability of the fermented product to bind to serum IgE in an allergic patient is comparable, thus indicating that although the soy protein is hydrolyzed into a small molecule peptide, there is still some sensitization to some extent. Bean protein or fragment.
  • the enzymatic method is to hydrolyze the antigenic protein in soybean by a specific enzyme, and the effect thereof is affected by various factors such as the type of the enzyme, the pretreatment method of hydrolysis, the degree of hydrolysis, and the like; in particular, there are various allergens in the soybean protein, and The surface antigenic determinant structure is complex, and how to simultaneously degrade various allergens to completely eliminate its sensitization is also a problem.
  • enzymatic hydrolysis can effectively destroy the epitope of soybean antigen protein, there are also concerns that some linear epitopes hidden in the three-dimensional structure of the protein or hydrophobic region may be exposed to make the enzymatic product have new allergenicity. .
  • the enzymatic degradation process also leads to the release of bitter and astringent ingredients in the soy protein, thereby affecting the taste and practical application of the product.
  • the invention provides a low sensitization and low bitterness soybean oligopeptide and a preparation method and application thereof, which are used for solving the technical defects that the sensitization of the soybean protein and the poor taste of the product cannot be completely eliminated in the prior art.
  • the preparation method of the low sensitization low bitterness soybean oligopeptide provided by the invention comprises the following steps:
  • soybean protein solution is thermally denatured to obtain a denatured protein solution
  • the protein content of the soybean protein powder used in the invention is >60%, further 60-95%; when preparing the soybean protein solution, the mass-to-volume ratio of the soy protein powder to water can be controlled to be 1: (5-10), That is, 1 kg of soy protein powder is mixed with 5 to 10 L of water to prepare a soy protein solution.
  • the concentration of the soy protein solution is too high (mass-volume ratio >1:5), the solution is relatively viscous, and its fluidity is poor, which tends to reduce the enzymatic hydrolysis efficiency; while the concentration is too low (mass-volume ratio ⁇ 1:10), the reaction volume is too large. Large, will affect the subsequent processing (such as membrane filtration, concentration, etc.), in addition to the corresponding increase in cost.
  • the thermal denaturation comprises: after heating the soy protein solution to 70-90 ° C, keeping warm and stirring for 20-60 min.
  • the heat denaturation treatment can destroy the spatial structure of the soy protein, thereby reducing the sensitization of the soy protein; at the same time, it can solve the problem that the soy protein solution has poor fluidity and the solution is thick, which is beneficial to the subsequent enzymatic hydrolysis.
  • the present inventors have conducted extensive research on the complete elimination of the sensitization of soy protein by the enzymatic method while suppressing the production of bitter and astringent substances in the enzymatic hydrolysate, and found that most proteases cannot completely eliminate the allergenicity of soy protein and / or inhibit the production of bitter and astringent substances in the enzymatic hydrolysate.
  • bromelain has no obvious effect on eliminating soy protein sensitization; neutral protease can eliminate the sensitization of soy protein to some extent, but bitter substances appear in the enzymatic product and cannot be removed.
  • the inventors unexpectedly discovered during the research that only the first enzymatic hydrolysis was first carried out using a complex enzyme consisting of a neutral protease and papain, followed by subsequent enzymatic hydrolysis using a complex enzyme consisting of an alkaline protease and a flavor protease (second Enzymatic hydrolysis can completely eliminate the sensitization of soy protein and inhibit the production of bitter and astringent substances in the enzymatic hydrolysate.
  • the amount of the neutral protease is 10 to 100 U/g
  • the amount of the papain is 10 to 100 U/g
  • the first enzymatic hydrolysis is between 30 and 60.
  • the temperature is °C
  • the time for controlling the first enzymatic hydrolysis is 1 to 3 hours.
  • the ratio of the amount of the neutral protease to the papain used is 1: (1 to 3).
  • the amount of the neutral protease is 10 U/g
  • the amount of papain is 10 to 30 U/g.
  • the use of a neutral protease in combination with papain is advantageous in controlling the release of the bitter and astringent component while sufficiently degrading the soy protein to eliminate its sensitization, and improving the mouthfeel of the hydrolyzed product.
  • the alkaline protease is used in an amount of 10 to 100 U/g
  • the flavor protease is used in an amount of 10 to 100 U/g
  • the second enzymatic solution is at a temperature of 30 to 60 ° C.
  • the next step is carried out, and the time for controlling the second enzymatic hydrolysis is 1 to 3 hours.
  • controlling the second enzymatic hydrolysis is carried out under the condition of pH 5-8, that is, if the pH of the first enzymatic hydrolysate is not in the range of 5-8, the pH of the first enzymatic hydrolyzate is adjusted to 5
  • alkaline protease and flavor protease are added for second enzymatic hydrolysis; and the ratio of the amount of alkaline protease to flavor protease used is 1: (1 to 4), for example, when the amount of alkaline protease is 10 U/g.
  • the amount of flavor protease is 10-40 U/g.
  • the short time ( ⁇ 1h) of the first enzymatic or second enzymatic reaction is not conducive to protein degradation, and too long (>3h) may lead to the production of bitter and astringent substances.
  • the two-step enzymatic hydrolysis facilitates the complete degradation of the soy protein into a lower molecular weight oligopeptide (for example, a peptide having a molecular weight of less than 1000 Da), thereby contributing to an increase in the utilization of soy protein.
  • a lower molecular weight oligopeptide for example, a peptide having a molecular weight of less than 1000 Da
  • the amount of each enzyme is based on the weight of the soy protein powder, that is, when a soy protein solution is prepared using 1 g of soy protein powder, a neutral protease of 10 to 100 U is used. Further, the inactivation of the enzyme is carried out at a temperature of 110 to 120 ° C, and the time for controlling the inactivation of the enzyme is 10 to 30 min.
  • the rotation speed of the centrifugation in the step 4) can be controlled to be 2000 to 6000 r/min, and the centrifugation can be performed by using conventional equipment such as a decanter centrifuge, a tube centrifuge, or the like.
  • the membrane filtration may be carried out using a membrane having a pore diameter of 1 to 200 nm, and the pore diameter may further be 1 to 50 nm; and when the membrane is filtered, the absolute pressure of the membrane filtration may be controlled to 0.2 to 0.4 MPa, and the temperature is 30 to 80 °C.
  • Membrane filtration of the centrifuged supernatant of the second enzymatic hydrolysate further removes components having a larger molecular weight, thereby maximally removing the macromolecular sensitizing protein component in the enzymatic hydrolysate.
  • the filtrate after filtration of the membrane, the filtrate can be decolored and concentrated. Specifically, it can be decolorized by a conventional decolorizing agent, for example, the decolorizing agent can be activated carbon powder, the mass ratio of the decolorizing agent to the filtrate can be (5-10):100, and the decoloring temperature can be controlled at 70-90 ° C, for example. At 80 ° C, the decolorization time can be 20 to 40 minutes, and the decolorization can be carried out under stirring. After decolorization, the decolorizing agent can be removed by conventional means such as filtration, such as plate and frame filtration.
  • the decolorizing agent can be activated carbon powder
  • the mass ratio of the decolorizing agent to the filtrate can be (5-10):100
  • the decoloring temperature can be controlled at 70-90 ° C, for example.
  • the decolorization time can be 20 to 40 minutes, and the decolorization can be carried out under stirring.
  • the decolorizing agent can be removed by conventional means such as filtration, such
  • the filtrate from which the decolorizing agent is removed may be evaporated to concentrate the filtrate, for example, a double effect falling film evaporator may be used for evaporation concentration, and the vapor pressure during evaporation may be controlled to be 0.1 ⁇ 0.02 MPa, and the evaporation temperature is 40 to 80° C.
  • concentration the volume of the concentrate can be reduced to 1/3 to 1/2 of the original volume.
  • sterilization and drying can be carried out, thereby producing a low-sensitization and low-bitter soybean oligopeptide powder, and drying can be, for example, spray drying.
  • the invention also provides a low sensitization and low bitterness soybean oligopeptide, which is prepared according to any one of the above preparation methods, wherein the content of glycoglobin in the hypoallergenic low bitter soy oligopeptide is ⁇ 200 mg/kg, ⁇ - the content of the concomitant globulin ⁇ 150 mg / kg, the content of the soybean trypsin inhibitor ⁇ 100 mg / kg; further, the content of the low sensitization and low bitter soy oligopeptide in the glycoglobulin ⁇ 125 mg / kg, ⁇ - The content of concomitant globulin is ⁇ 90 mg/kg, and the content of soybean trypsin inhibitor is ⁇ 50 mg/kg.
  • the low-sensitivity low bitterness soybean oligopeptide has a molecular weight of less than 5000 Da and a mass content of >85%, and a peptide having a molecular weight of less than 1000 Da has a mass content of >60%; further, the low sensitization and low bitterness
  • the mass content of the peptide having a molecular weight of less than 5000 Da in the soybean oligopeptide is >95%, and the mass content of the peptide having a molecular weight of less than 1000 Da is >85%.
  • the invention also provides the use of the above low sensitization low bitter taste soybean oligopeptide in milk powder or health food.
  • the milk powder may include infant milk powder, adult milk powder, middle-aged milk powder and the like.
  • the method of the invention utilizes four specific proteases to perform enzymatic hydrolysis in two steps after heat denaturation of soybean protein, which not only overcomes the variety of soy protein allergens and the complex structure of surface antigenic determinants, and cannot completely eliminate the sensitizing effect thereof.
  • the problem is that the total content of the main sensitizing proteins of soy protein, glycinin, ⁇ -conglycinin and soybean trypsin inhibitor, is reduced by more than 99%; in addition, the method avoids the release of bitter and astringent ingredients in the soy protein. Thereby ensuring the taste of the product.
  • the method of the invention has simple process and is suitable for large-scale production, and the prepared low-sensitivity low-bitter soybean oligopeptide is widely used.
  • Each protease used in the present invention was purchased from Novozymes Biotechnology Co., Ltd.
  • the pH value is adjusted to about 7, and neutral protease and papain are added to the denatured protein solution, wherein the amount of neutral protease and papain is 50 U/g soybean.
  • the first enzymatic hydrolysis was carried out at a temperature of about 50 ° C, and the first enzymatic hydrolyzate was prepared after the first enzymatic hydrolysis for about 3 hours.
  • the alkaline protease and the flavor protease are continuously added to the first enzymatic hydrolysate, wherein the amount of the alkaline protease is about 50 U/g of soy protein powder, and the amount of the flavor protease is about 100 U/g of soy protein powder, which is maintained at about 50 ° C.
  • the second enzymatic hydrolysis was carried out at a temperature, and after the second enzymatic hydrolysis for about 2 hours, the enzymatic hydrolysate was heated to 120 ° C for 20 min to obtain a second enzymatic hydrolysate.
  • the second enzymatic hydrolysate was centrifuged at a speed of 4000 r/min, and the supernatant was collected for use;
  • the centrifugal supernatant was filtered using a ceramic membrane having a pore diameter of about 50 nm, and the absolute pressure of the filtration was controlled to be about 0.3 MPa, and the temperature was about 50 ° C to obtain a filtrate.
  • the activated carbon powder is added to the filtrate, and the mass ratio of the activated carbon powder to the filtrate is 10:100, and the mixture is stirred at a temperature of about 80 ° C for about 30 minutes for decolorization, and after decolorization, the plate is filtered to remove the activated carbon powder to obtain a decolorizing solution;
  • the Glycincin ELISA Kit (Unibiotest) and the ⁇ -conglycinin ELISA Kit (Unibiotest) were used to detect the content of glycinin and ⁇ -conglycinin in low-sensitization and low-bitter soybean oligopeptides, respectively, using Soy Allergens kit (ELISA SYSTEM)
  • ELISA SYSTEM Soy Allergens kit
  • the content of soybean trypsin inhibitor in low sensitization and low bitterness soybean oligopeptide was detected, and the soybean protein solution without any treatment was used as a blank control.
  • the quality test results are shown in Table 1.
  • the low-sensitization and low-bitter soybean oligopeptide prepared above was dissolved in water to prepare a low-sensitivity low-bitter soybean oligopeptide solution having a mass content of 10%; the tissue 20-person evaluation group (men and women) was hypoallergenic
  • the bitter taste evaluation of the low bitter soy oligopeptide solution was carried out by taking 1 mL of the low sensitization low bitter soy oligopeptide solution, and gradually diluting it to just taste the bitterness, and calculating the dilution factor as the bitterness value, and calculating 20
  • the average bitterness value of humans is shown in Table 3.
  • the pH value is adjusted to about 8, and a neutral protease and papain are added to the denatured protein solution, wherein the amount of the neutral protease is about 10 U/g of soy protein powder.
  • the amount of papain was about 30 U/g of soy protein powder, and the first enzymatic hydrolysis was carried out at a temperature of about 40 ° C. After the first enzymatic hydrolysis for about 2 h, the first enzymatic hydrolysate was prepared.
  • the alkaline protease and the flavor protease are continuously added to the first enzymatic hydrolysate, wherein the amount of the alkaline protease and the flavor protease is about 75 U/g of the soy protein powder, and the second enzymatic hydrolysis is carried out at a temperature of about 40 ° C. After the second enzymatic hydrolysis for about 3 hours, the enzymatic hydrolysate was heated to 110 ° C for 30 min to obtain a second enzymatic hydrolysate.
  • the second enzymatic hydrolysate was centrifuged at a rotational speed of 3500 r/min, and the centrifugation supernatant was collected for use;
  • the supernatant is filtered by a filter with a pore size of about 200 nm to control the absolute pressure of the filtration.
  • the filtrate was obtained at about 0.4 MPa and at a temperature of about 80 °C.
  • the activated carbon powder is added to the filtrate, and the mass ratio of the activated carbon powder to the filtrate is 5:100, and the mixture is stirred at a temperature of about 80 ° C for about 30 minutes for decolorization, and after decolorization, the plate is filtered to remove the activated carbon powder to obtain a decolorizing liquid;
  • the decolorizing liquid is subjected to evaporation concentration, the vapor pressure is controlled to be about 0.1 MPa, the evaporation temperature is about 80 ° C, and the decolorizing liquid is concentrated to 1/3 of the original volume, and the concentrated liquid is sterilized and spray dried to obtain low sensitization.
  • Low bitter taste soybean oligopeptide the quality test results, molecular weight distribution and mouthfeel evaluation results of the low sensitization and low bitterness soybean oligopeptide are shown in Tables 1 to 3, respectively.
  • the pH value is adjusted to about 6, and a neutral protease and papain are added to the denatured protein solution, wherein the amount of the neutral protease is about 50 U/g of soy protein powder.
  • the amount of papain used is about 100 U/g of soy protein powder, and the first enzymatic hydrolysis is carried out at a temperature of about 60 ° C. After the first enzymatic hydrolysis for about 1 h, the first enzymatic hydrolysate is prepared.
  • the alkaline protease and the flavor protease are continuously added to the first enzymatic hydrolysate, wherein the amount of the alkaline protease is about 40 U/g of soy protein powder, and the amount of the flavor protease is about 160 U/g of soy protein powder, which is maintained at about 60 ° C.
  • the second enzymatic hydrolysis was carried out at a temperature, and after the second enzymatic hydrolysis for about 1 hour, the enzymatic hydrolysate was heated to 120 ° C for 20 min to obtain a second enzymatic hydrolysate.
  • the second enzymatic hydrolysate was centrifuged at a speed of 4000 r/min, and the supernatant was collected for use;
  • the supernatant of the centrifuged membrane was subjected to membrane filtration using a membrane having a pore size of about 50 nm, and the absolute pressure of the membrane filtration was controlled to be about 0.2 MPa, and the temperature was about 30 ° C to obtain a filtrate.
  • the activated carbon powder is added to the filtrate, and the mass ratio of the activated carbon powder to the filtrate is 8:100, and the temperature is about 80 ° C.
  • the mixture was stirred for about 30 minutes for decolorization, and after decolorization, the plate frame was filtered to remove the activated carbon powder to obtain a decolorizing liquid;
  • the decolorizing liquid is subjected to evaporation concentration, the vapor pressure is controlled to be about 0.1 MPa, the evaporation temperature is about 60 ° C, and the decolorizing liquid is concentrated to 1/3 of the original volume, and the concentrated liquid is sterilized and spray-dried to obtain low sensitization.
  • Low bitter taste soybean oligopeptide the quality test results, molecular weight distribution and mouthfeel evaluation results of the low sensitization and low bitterness soybean oligopeptide are shown in Tables 1 to 3, respectively.
  • Example 2 After the denatured protein solution prepared in Example 1 was lowered to about 40 ° C, the pH was adjusted to about 8, and a neutral protease was added to the denatured protein solution according to the amount of 100 U/g of soy protein powder to maintain a temperature of about 40 ° C. After enzymatic hydrolysis for about 5 hours, the enzymatic hydrolysate was sequentially centrifuged, concentrated, sterilized, and dried according to the method of Example 1, to obtain a soybean peptide. The quality test results and the taste evaluation results are shown in Tables 1 and 3, respectively.
  • Example 1 After the denatured protein solution prepared in Example 1 was lowered to about 50 ° C, the pH was adjusted to about 7, and bromelain was added to the denatured protein solution according to the dosage of 250 U/g of soy protein powder, and maintained at a temperature of about 50 ° C. After enzymatic hydrolysis for about 5 hours, the enzymatic hydrolysate was sequentially centrifuged, concentrated, sterilized, and dried according to the method of Example 1, to obtain a soybean peptide. The quality test results and the taste evaluation results are shown in Tables 1 and 3, respectively.
  • the second enzymatic hydrolysate prepared in Example 1 was directly centrifuged, concentrated, sterilized and dried according to the method of Example 1 without membrane filtration and decolorization to obtain soybean peptide, and the quality test results and the taste evaluation results were respectively See Tables 1 and 3.
  • the content of the sensitizing protein glycinin, ⁇ -conglycinin and soybean trypsin inhibitor is significantly reduced in the low sensitization and low bitterness soybean oligopeptide prepared by the invention, and the total mass content of the three proteins can be reduced by 99. Above %, the method of the present invention can completely eliminate the sensitization of soy protein, and the desensitization effect is good.
  • bromelain to treat soy protein to eliminate the soy protein sensitization effect is not obvious; the use of neutral protease to soy protein can eliminate the sensitization of soy protein to a certain extent, but the desensitization effect is generally .
  • the allergen protein component of soybean can not be completely eliminated only by the enzymatic hydrolysis process, and only the soybean membrane allergen can be eliminated to the greatest extent by combining the specific membrane filtration and decolorization processes on the basis of the composite enzymatic hydrolysis process of the present invention.
  • proteases or combinations thereof can reduce or eliminate the sensitization of soy protein when the soy protein is treated, and only adopt a specific composition of protease and adopt a specific process (for example, pre-denaturation, step-by-step enzymatic hydrolysis, Membrane filtration, decolorization and other processes can completely eliminate the sensitization of soy protein.
  • Example 1 Example 2
  • Example 3 More than 5000 2.34 1.13 2.23 1000-5000 5.20 10.24 9.69 500-1000 22.78 27.02 29.64 140-500 65.32 56.51 54.85 Below 140 4.22 5.09 3.49 Below 5000 97.66 98.87 97.77 Below 1000 92.32 88.62 87.98
  • the low sensitization low bitterness soybean oligopeptide prepared by the invention has a mass content of less than 5000 Da and a mass content of >95%, and a peptide having a molecular weight of less than 1000 Da has a mass content of >85%.
  • the low sensitization and low bitterness soybean oligopeptide prepared by the invention has less bitterness component and good mouthfeel, indicating that the method of the invention can effectively inhibit the production of bitter substances in the enzymatic hydrolysate; and the proteases such as bromelain and neutral protease are used for soybean.
  • the proteases such as bromelain and neutral protease are used for soybean.

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Abstract

本发明提供一种低致敏低苦味大豆低聚肽及其制备方法和应用。该制备方法包括：1）将大豆蛋白溶液进行热变性；2）调节pH值至6-9后，加入中性蛋白酶和木瓜蛋白酶进行第一酶解；3）加入碱性蛋白酶和风味蛋白酶进行第二酶解；4）离心，膜过滤，得到低致敏低苦味大豆低聚肽。

Description

一种低致敏低苦味大豆低聚肽及其制备方法和应用 技术领域
本发明涉及一种大豆低聚肽，特别是涉及一种低致敏低苦味大豆低聚肽及其制备方法和应用。
背景技术
大豆蛋白是一种植物性蛋白，其氨基酸组成与牛奶蛋白相近，各种必需氨基酸含量均较为丰富，在营养价值上可与动物蛋白等同，在基因结构上也最接近人体氨基酸，是最具营养的植物蛋白。然而，大豆蛋白中存在多种致敏原，例如大豆球蛋白、β-伴球蛋白、P34、GlymBd 28K等，其中大豆球蛋白和β-伴球蛋白是大豆中蛋白质的主要构成成分，二者约占到70％；而有些大豆蛋白例如大豆胰蛋白酶抑制剂(STI)在常规生产过程中(例如高温条件下)结构依然保持稳定，因此常常被用作检测大豆过敏蛋白的指示剂。目前，大约有1～6％的婴儿会受到大豆致敏原的影响而产生呼吸、皮肤、胃肠道症状等大豆过敏反应，并且随着大豆产品越来越多，成年人大豆过敏的发病率也在不断上升。
对大豆蛋白进行脱敏的方法包括热处理、化学处理、发酵法、酶法等。热处理是最常用的大豆致敏原脱敏方法，其能改变大豆蛋白的结构并降低抗原蛋白的致敏活性，然而由于P34蛋白中表面抗原决定簇结构的复杂性，不可能仅依靠加热使蛋白质变性而较为彻底地消除其致敏作用。化学处理主要是利用化学试剂来降低胰蛋白酶抑制剂的活性，然而其不可避免地会产生化学残留等食品安全问题。
发酵法主要利用霉菌、枯草芽孢杆菌等微生物来降解大豆制品中的抗原蛋白，尽管发酵可将大豆蛋白水解成致敏性较低的小分子肽，然而水解蛋白中是否保留被抗体识别的必要构象仍成问题。例如公开号为CN101990984A的专利公开了一种饲用高抗氧化低致敏性发酵豆粕制备方法，采用米曲霉对豆粕发酵基料进行发酵，尽管发酵后大分子蛋白被明显降解，然而其并未对发酵产品的致敏性进行检测，因此无法确定发酵产品中是否仍然存在致敏大豆片段；此外该方法没有对发酵产品的口感进行评价。Herian等人用放射变应原吸附测定法(RAST)检测了五种传统的大豆发酵制品的致敏性，其中包括豆芽、酸水解酱油、霉菌水解酱油、豆豉和豆酱，结果表明五种大豆发酵制品与过敏患者血清IgE结合的能力相当，由此说明尽管大豆蛋白被水解为小分子肽，然而在某种程度上仍然存在致敏大 豆蛋白或片段。
酶法是通过特定的酶来水解大豆中的抗原蛋白，其作用效果受到酶的种类、水解前处理方式、水解程度等诸多因素的影响；特别是，大豆蛋白中存在多种致敏原，并且其表面抗原决定簇结构复杂，如何能够同时对多种致敏原进行降解以彻底地消除其致敏作用也成问题。此外，酶水解虽然可有效破坏大豆抗原蛋白的抗原表位，但也存在使一些隐藏在蛋白三维结构内部或疏水区的线性抗原表位暴露出来而使酶解产物具有新的致敏性的担忧。同时，酶法降解过程中还会导致大豆蛋白中的苦味涩味成分释放，从而影响产品的口感和实际应用。
发明内容
本发明提供一种低致敏低苦味大豆低聚肽及其制备方法和应用，用于解决现有技术中无法彻底消除大豆蛋白的致敏性以及产品口感不佳等技术缺陷。
本发明提供的低致敏低苦味大豆低聚肽的制备方法，包括如下步骤：
1)将大豆蛋白粉与水混合制成大豆蛋白溶液后，对大豆蛋白溶液进行热变性，制得变性蛋白溶液；
2)调节所述变性蛋白溶液的pH值至6～9后，加入中性蛋白酶和木瓜蛋白酶进行第一酶解，制得第一酶解液；
3)向所述第一酶解液中加入碱性蛋白酶和风味蛋白酶进行第二酶解，灭酶后，制得第二酶解液；
4)将所述第二酶解液离心后，对离心上清液进行膜过滤，制得低致敏低苦味大豆低聚肽。
本发明采用的大豆蛋白粉中蛋白质的质量含量＞60％，进一步为60～95％；在制备大豆蛋白溶液时，可控制大豆蛋白粉与水的质量体积比为1：(5～10)，即：1kg的大豆蛋白粉与5～10L的水进行混合制备大豆蛋白溶液。大豆蛋白溶液的浓度过高(质量体积比＞1：5)时溶液较为粘稠，其流动性差，易导致酶解效率降低；而浓度过低(质量体积比＜1：10)时反应体积过大，会影响后续处理(例如膜过滤、浓缩等)，此外成本也会相应增加。
进一步地，所述热变性包括：将大豆蛋白溶液加热至70～90℃后，保温并持续搅拌20～60min。该热变性处理能够破坏大豆蛋白的空间结构，从而降低大豆蛋白的致敏性；同时还可解决大豆蛋白溶液流动性差，溶液粘稠的问题，有利于后续酶解的进行。
本发明人对于采用酶法彻底消除大豆蛋白的致敏性同时抑制酶解产物中苦味涩味物质的产生做了大量的研究，结果发现大多数的蛋白酶无法较为彻底消除大豆蛋白的致敏性和/或抑制酶解产物中苦味涩味物质的产生。例如，菠萝蛋白酶对消除大豆蛋白致敏性作用不明显；中性蛋白酶在一定程度上能够消除大豆蛋白的致敏性，然而酶解产物中出现苦味物质并且无法去除。发明人在研究过程中意外地发现，只有首先采用由中性蛋白酶和木瓜蛋白酶组成的复合酶进行第一酶解，随后采用由碱性蛋白酶和风味蛋白酶组成的复合酶进行后续酶解(第二酶解)才能够较为彻底地消除大豆蛋白的致敏性同时抑制酶解产物中苦味涩味物质的产生。
特别是，在本发明的第一酶解中，所述中性蛋白酶的用量为10～100U/g，所述木瓜蛋白酶的用量为10～100U/g，所述第一酶解在30～60℃的温度下进行，并且控制第一酶解的时间为1～3h。进一步地，所使用的中性蛋白酶与木瓜蛋白酶的用量比为1：(1～3)，例如中性蛋白酶的用量为10U/g时，木瓜蛋白酶的用量为10～30U/g。中性蛋白酶与木瓜蛋白酶的结合使用有利于在充分降解大豆蛋白以消除其致敏性的同时控制苦味涩味成分的释放，并改善酶解产物的口感。
在本发明的第二酶解中，所述碱性蛋白酶的用量为10～100U/g，所述风味蛋白酶的用量为10～100U/g，所述第二酶解在30～60℃的温度下进行，并且控制第二酶解的时间为1～3h。进一步地，控制第二酶解在pH值为5～8的条件下进行，即，如果第一酶解液的pH值不在5～8的范围内，调节第一酶解液的pH值至5～8后加入碱性蛋白酶和风味蛋白酶进行第二酶解；并且，所使用的碱性蛋白酶与风味蛋白酶的用量比为1：(1～4)，例如碱性蛋白酶的用量为10U/g时，风味蛋白酶的用量为10～40U/g。第一酶解或第二酶解的时间过短(＜1h)均不利于蛋白的降解，而时间过长(＞3h)可能导致苦味涩味物质的产生。
在第一酶解后同时采用碱性蛋白酶和风味蛋白酶继续进行酶解有利于进一步对第一酶解产物进行降解以消除大豆蛋白的致敏性，同时可控制苦味涩味成分的释放并改善酶解产物口感，两步酶解可将大豆蛋白中的主要致敏蛋白(包括大豆球蛋白和β-伴球蛋白)以及胰蛋白酶抑制剂的总含量降低99％以上。此外，两步酶解有利于将大豆蛋白充分降解为分子量较小的低聚肽(例如分子量小于1000Da的肽)，从而有利于提高大豆蛋白的利用率。
在本发明中，各酶的用量是基于大豆蛋白粉的重量，即，在采用1g大豆蛋白粉制备大豆蛋白溶液时，使用10～100U的中性蛋白酶。进一步地，在110～120℃的温度下进行所述灭酶，并且控制灭酶的时间为10～30min。
进一步地，可控制步骤4)中所述离心的转速为2000～6000r/min，离心可采用常规设备进行，例如卧螺离心机、管式离心机等。此外，可采用孔径为1～200nm的滤膜进行所述膜过滤，孔径进一步可为1～50nm；膜过滤时，可控制膜过滤的绝对压力为0.2～0.4MPa，温度为30～80℃。对第二酶解液的离心上清液进行膜过滤，可进一步地截留分子量较大的成分，从而最大限度地去除酶解液中的大分子致敏蛋白组分。
在本发明中，在膜过滤后，可对滤液进行脱色和浓缩。具体地，可以采用常规脱色剂进行脱色，脱色剂例如可以为活性碳粉，脱色剂与滤液的质量配比可以为(5～10)：100，脱色的温度可控制在70～90℃，例如80℃，脱色时间可以为20～40min，脱色可在搅拌下进行。在脱色后，可通过过滤等常规方式去除脱色剂，例如板框过滤。进一步地，可对去除脱色剂的滤液进行蒸发以浓缩滤液，例如可采用双效降膜蒸发器进行蒸发浓缩，并且可控制蒸发时的蒸汽压为0.1±0.02MPa，蒸发温度为40～80℃，经浓缩后，浓缩液的体积可降至原体积的1/3～1/2。进一步地，在浓缩后可进行灭菌和干燥，从而制得低致敏低苦味大豆低聚肽粉，干燥例如可以为喷雾干燥。
本发明还提供一种低致敏低苦味大豆低聚肽，按照上述任一所述制备方法制得，所述低致敏低苦味大豆低聚肽中大豆球蛋白的含量＜200mg/kg，β-伴球蛋白的含量＜150mg/kg，大豆胰蛋白酶抑制剂的含量＜100mg/kg；进一步地，所述低致敏低苦味大豆低聚肽中大豆球蛋白的含量＜125mg/kg，β-伴球蛋白的含量＜90mg/kg，大豆胰蛋白酶抑制剂的含量＜50mg/kg。
进一步地，所述低致敏低苦味大豆低聚肽中分子量小于5000Da的肽的质量含量＞85％，分子量小于1000Da的肽的质量含量＞60％；更进一步地，所述低致敏低苦味大豆低聚肽中分子量小于5000Da的肽的质量含量＞95％，分子量小于1000Da的肽的质量含量＞85％。
本发明还提供上述低致敏低苦味大豆低聚肽在奶粉或保健食品中的应用。其中，所述奶粉可包括婴幼儿奶粉、成人奶粉、中老年奶粉等。
本发明的方法在对大豆蛋白热变性后利用四种特定的蛋白酶分两步进行酶解，不仅克服了大豆蛋白致敏原种类多且表面抗原决定簇结构复杂而无法较为彻底消除其致敏作用的问题，大豆蛋白的主要致敏蛋白大豆球蛋白、β-伴球蛋白和大豆胰蛋白酶抑制剂的总含量降低99％以上；此外，该方法避免了大豆蛋白中的苦味涩味成分的释放，从而保证了产品的口感。本发明的方法工艺简单，适合大规模生产，所制得的低致敏低苦味大豆低聚肽应用范围广泛。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明采用的各蛋白酶均购自诺维信生物技术有限公司。
实施例1
1、热变性
将500kg蛋白含量为60％左右的大豆蛋白粉加入反应罐后，向反应罐中加入4000L水，搅拌混匀制成大豆蛋白溶液后，将大豆蛋白溶液加热至80℃左右，保温并持续搅拌约40min，制得变性蛋白溶液。
2、第一酶解
待上述变性蛋白溶液的温度降至50℃左右后，调节其pH值至7左右，向变性蛋白溶液中加入中性蛋白酶和木瓜蛋白酶，其中中性蛋白酶和木瓜蛋白酶的用量均为50U/g大豆蛋白粉左右，维持在50℃左右的温度下进行第一酶解，第一酶解约3h后，制得第一酶解液。
3、第二酶解
向上述第一酶解液中继续加入碱性蛋白酶和风味蛋白酶，其中碱性蛋白酶的用量为50U/g大豆蛋白粉左右，风味蛋白酶的用量为100U/g大豆蛋白粉左右，维持在50℃左右的温度下进行第二酶解，第二酶解约2h后，将酶解液加热至120℃灭酶20min，制得第二酶解液。
4、离心、膜过滤
在4000r/min的转速下对第二酶解液进行离心，收集离心上清液备用；
采用孔径为50nm左右的陶瓷膜对离心上清液进行过滤，控制过滤的绝对压力为0.3MPa左右，温度50℃左右，得到滤液。
5、脱色、浓缩、灭菌
向滤液中加入活性碳粉，活性碳粉与滤液的质量配比为10：100，在80℃左右的温度下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为60℃左右，使脱色 液浓缩至原体积的1/2后，浓缩液经灭菌、喷雾干燥后，制得低致敏低苦味大豆低聚肽。
6、质量检测及口感评价
采用Glycincin ELISA Kit(Unibiotest公司)和β-conglycinin ELISA Kit(Unibiotest公司)分别检测低致敏低苦味大豆低聚肽中大豆球蛋白和β-伴球蛋白的含量，采用Soy Allergens试剂盒(ELISASYSTEM公司)检测低致敏低苦味大豆低聚肽中大豆胰蛋白酶抑制剂的含量，同时以未经任何处理的大豆蛋白溶液作为空白对照，质量检测结果见表1。
参照GB/T 22729-2008对上述制备的低致敏低苦味大豆低聚肽中各组分的分子量分布进行检测，结果见表2。
将上述制备的低致敏低苦味大豆低聚肽溶于水中，制成质量含量为10％的低致敏低苦味大豆低聚肽溶液；组织20人评定小组(男女各半)对低致敏低苦味大豆低聚肽溶液进行苦味评价，评价方法为：取1mL低致敏低苦味大豆低聚肽溶液，对其进行梯度稀释至刚好尝出苦味为止，将稀释倍数计为苦味值，计算20人的平均苦味值，结果见表3。
实施例2
1、热变性
将500kg蛋白含量为65％左右的大豆蛋白粉加入反应罐后，向反应罐中加入5000L水，搅拌混匀制成大豆蛋白溶液后，将大豆蛋白溶液加热至90℃左右，保温并持续搅拌约20min，制得变性蛋白溶液。
2、第一酶解
待上述变性蛋白溶液的温度降至40℃左右后，调节其pH值至8左右，向变性蛋白溶液中加入中性蛋白酶和木瓜蛋白酶，其中中性蛋白酶的用量为10U/g大豆蛋白粉左右，木瓜蛋白酶的用量为30U/g大豆蛋白粉左右，维持在40℃左右的温度下进行第一酶解，第一酶解约2h后，制得第一酶解液。
3、第二酶解
向上述第一酶解液中继续加入碱性蛋白酶和风味蛋白酶，其中碱性蛋白酶和风味蛋白酶的用量均为75U/g大豆蛋白粉左右，维持在40℃左右的温度下进行第二酶解，第二酶解约3h后，将酶解液加热至110℃灭酶30min，制得第二酶解液。
4、离心、膜过滤
在3500r/min的转速下对第二酶解液进行离心，收集离心上清液备用；
采用孔径为200nm左右的滤膜对离心上清液进行过滤，控制过滤的绝对压力为 0.4MPa左右，温度80℃左右，得到滤液。
5、脱色、浓缩、灭菌
向滤液中加入活性碳粉，活性碳粉与滤液的质量配比为5：100，在80℃左右的温度下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为80℃左右，使脱色液浓缩至原体积的1/3后，浓缩液经灭菌、喷雾干燥后，制得低致敏低苦味大豆低聚肽；该低致敏低苦味大豆低聚肽的质量检测结果、分子量分布和口感评价结果分别见表1至表3。
实施例3
1、热变性
将500kg蛋白含量为70％左右的大豆蛋白粉加入反应罐后，向反应罐中加入2500L水，搅拌混匀制成大豆蛋白溶液后，将大豆蛋白溶液加热至70℃左右，保温并持续搅拌约60min，制得变性蛋白溶液。
2、第一酶解
待上述变性蛋白溶液的温度降至60℃左右后，调节其pH值至6左右，向变性蛋白溶液中加入中性蛋白酶和木瓜蛋白酶，其中中性蛋白酶的用量为50U/g大豆蛋白粉左右，木瓜蛋白酶的用量为100U/g大豆蛋白粉左右，维持在60℃左右的温度下进行第一酶解，第一酶解约1h后，制得第一酶解液。
3、第二酶解
向上述第一酶解液中继续加入碱性蛋白酶和风味蛋白酶，其中碱性蛋白酶的用量为40U/g大豆蛋白粉左右，风味蛋白酶的用量为160U/g大豆蛋白粉左右，维持在60℃左右的温度下进行第二酶解，第二酶解约1h后，将酶解液加热至120℃灭酶20min，制得第二酶解液。
4、离心、膜过滤
在4000r/min的转速下对第二酶解液进行离心，收集离心上清液备用；
采用孔径为50nm左右的滤膜对离心上清液进行膜过滤，控制膜过滤的绝对压力为0.2MPa左右，温度30℃左右，得到滤液。
5、脱色、浓缩、灭菌
向滤液中加入活性碳粉，活性碳粉与滤液的质量配比为8：100，在80℃左右的温度 下搅拌30min左右进行脱色，脱色后板框过滤去除活性碳粉，得到脱色液；
对脱色液进行蒸发浓缩，控制蒸汽压为0.1MPa左右，蒸发温度为60℃左右，使脱色液浓缩至原体积的1/3后，浓缩液经灭菌、喷雾干燥后，制得低致敏低苦味大豆低聚肽；该低致敏低苦味大豆低聚肽的质量检测结果、分子量分布和口感评价结果分别见表1至表3。
对照例1
将实施例1制备的变性蛋白溶液降至40℃左右后，调节其pH值至8左右，按照用量100U/g大豆蛋白粉左右向变性蛋白溶液中加入中性蛋白酶，维持在40℃左右的温度下酶解约5h后，将酶解液按照实施例1方法依次进行离心、浓缩、灭菌、干燥，制得大豆肽，其质量检测结果和口感评价结果分别见表1和表3。
对照例2
将实施例1制备的变性蛋白溶液降至50℃左右后，调节其pH值至7左右，按照用量250U/g大豆蛋白粉左右向变性蛋白溶液中加入菠萝蛋白酶，维持在50℃左右的温度下酶解约5h后，将酶解液按照实施例1方法依次进行离心、浓缩、灭菌、干燥，制得大豆肽，其质量检测结果和口感评价结果分别见表1和表3。
对照例3
将实施例1制备的第二酶解液，不经过膜过滤和脱色而直接按照实施例1方法依次进行离心、浓缩、灭菌、干燥，制得大豆肽，其质量检测结果和口感评价结果分别见表1和表3。
表1各大豆肽的质量检测结果
Figure PCTCN2015078137-appb-000001
Figure PCTCN2015078137-appb-000002
由表1结果可知：
1、本发明制备的低致敏低苦味大豆低聚肽中，致敏蛋白大豆球蛋白、β-伴球蛋白以及大豆胰蛋白酶抑制剂的含量显著降低，三种蛋白的总质量含量可降低99％以上，说明本发明方法能够较为彻底地消除大豆蛋白的致敏性，脱敏效果良好。
2、采用菠萝蛋白酶对大豆蛋白进行处理时消除大豆蛋白致敏性作用不明显；采用中性蛋白酶对大豆蛋白进行处理时能够在一定程度上能够消除大豆蛋白的致敏性，但脱敏效果一般。
3、仅通过酶解工艺无法完全消除大豆的过敏原蛋白组分，而只有在本发明的复合酶解工艺的基础上结合特定的膜过滤以及脱色等工艺才能够最大限度地消除大豆过敏原。
由此说明：并非任意的蛋白酶或其组合对大豆蛋白进行处理时均可降低或消除大豆蛋白的致敏性，而只有采用特定组成的蛋白酶并采用特定工艺(例如预变性、分步酶解、膜过滤、脱色等工艺)才能够较为彻底地消除大豆蛋白的致敏性。
表2低致敏低苦味大豆低聚肽的分子量分布
分子量范围 实施例1 实施例2 实施例3
5000以上 2.34 1.13 2.23
1000-5000 5.20 10.24 9.69
500-1000 22.78 27.02 29.64
140-500 65.32 56.51 54.85
140以下 4.22 5.09 3.49
5000以下 97.66 98.87 97.77
1000以下 92.32 88.62 87.98
由表2结果可知：
本发明制备的低致敏低苦味大豆低聚肽中分子量小于5000Da的肽的质量含量＞95％，分子量小于1000Da的肽的质量含量＞85％。
表3各大豆肽口感评价结果
试验例 平均苦味值
实施例1 3
实施例2 2
实施例3 2
对照例1 6
对照例2 7
对照例3 6
由表3结果可知：
本发明制备的低致敏低苦味大豆低聚肽中苦味成分少，口感较好，说明本发明方法能够有效抑制酶解产物中苦味物质的产生；而采用菠萝蛋白酶、中性蛋白酶等蛋白酶对大豆蛋白进行处理时无法有效避免大豆蛋白中的苦味涩味成分的释放。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. 一种低致敏低苦味大豆低聚肽的制备方法，其特征在于，包括如下步骤：
    1)将大豆蛋白粉与水混合制成大豆蛋白溶液后，对大豆蛋白溶液进行热变性，制得变性蛋白溶液；
    2)调节所述变性蛋白溶液的pH值至6～9后，加入中性蛋白酶和木瓜蛋白酶进行第一酶解，制得第一酶解液；
    3)向所述第一酶解液中加入碱性蛋白酶和风味蛋白酶进行第二酶解，灭酶后，制得第二酶解液；
    4)将所述第二酶解液离心后，对离心上清液进行膜过滤，制得低致敏低苦味大豆低聚肽。
  2. 根据权利要求1所述的制备方法，其特征在于，所述大豆蛋白粉与水的质量体积比为1：(5～10)。
  3. 根据权利要求1所述的制备方法，其特征在于，所述热变性包括：将大豆蛋白溶液加热至70～90℃后，保温并持续搅拌20～60min。
  4. 根据权利要求1所述的制备方法，其特征在于，所述中性蛋白酶的用量为10～100U/g，所述木瓜蛋白酶的用量为10～100U/g，所述第一酶解在30～60℃的温度下进行，并且控制第一酶解的时间为1～3h。
  5. 根据权利要求1所述的制备方法，其特征在于，所述碱性蛋白酶的用量为10～100U/g，所述风味蛋白酶的用量为10～100U/g，所述第二酶解在30～60℃的温度下进行，并且控制第二酶解的时间为1～3h。
  6. 根据权利要求1所述的制备方法，其特征在于，在110～120℃的温度下进行所述灭酶，并且控制灭酶的时间为10～30min。
  7. 根据权利要求1所述的制备方法，其特征在于，采用孔径为1～200nm的滤膜进行所述膜过滤。
  8. 一种低致敏低苦味大豆低聚肽，其特征在于，按照权利要求1至7中任一所述制备方法制得，所述低致敏低苦味大豆低聚肽中大豆球蛋白的含量＜200mg/kg，β-伴球蛋白的含量＜150mg/kg，大豆胰蛋白酶抑制剂的含量＜100mg/kg。
  9. 根据权利要求8所述的低致敏低苦味大豆低聚肽，其特征在于，所述低致敏低苦味大豆低聚肽中分子量小于5000Da的肽的质量含量＞85％，分子量小于1000Da的肽的质量含量＞60％。
  10. 权利要求8或9所述的低致敏低苦味大豆低聚肽在奶粉或保健食品中的应用。
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CN104286856A (zh) * 2014-10-20 2015-01-21 长春大学 无异味、高纯度大豆低聚肽的生产方法

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CN107624952A (zh) * 2017-11-07 2018-01-26 山东禹王生态食业有限公司 一种大豆蛋白酶解***及方法
NL2029200A (en) * 2020-10-26 2022-06-16 Dalian Hongrun Lianhua Food Co Ltd Gluten-free hypoallergenic soy protein flour and preparation method thereof
CN113234786A (zh) * 2021-06-17 2021-08-10 广西壮族自治区农业科学院 一种从富硒稻米加工副产物中提取富硒多肽的方法
CN115720956A (zh) * 2022-12-01 2023-03-03 无锡金农生物科技有限公司 一种高悬浮稳定性、低苦味大米蛋白的生产方法
CN117987499A (zh) * 2024-01-25 2024-05-07 清远市望莎生物科技有限公司 一种多肽组合物及其制备方法、应用、日化品

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